ES2259191T3 - METHOD FOR EXPRESSING FORANE GENES AND VECTORS FOR IT. - Google Patents

Abstract

A method for expressing a foreign gene comprises inserting said foreign gene into a site downstream of a promoter, and expressing said foreign gene in a cell, characterized in that the sequence shown in SEQ ID NO: 3 in the SEQUENCE LISTING or a functional variant thereof is inserted into a site upstream of said foreign gene, and said foreign gene is expressed.A method for expressing a foreign gene comprises inserting said foreign gene into a site downstream of a promoter, and expressing said foreign gene in a cell, characterized in that the sequence shown in SEQ ID NO: 3 in the SEQUENCE LISTING or a functional variant thereof is inserted into a site upstream of said foreign gene, and said foreign gene is expressed.

Description

Method to express foreign genes and vectors for it.

Technical scope

The present invention relates to a method to express a foreign gene and a vector for it. More in In particular, the present invention relates to a method for express a foreign gene in genetic engineering processes, being with this method the expression of the foreign gene promoted in greater as in the case of conventional methods, and to a vector for it.

Prior art

The promotion of foreign gene expression It is one of the most necessary techniques in engineering processes genetics, especially when genetic engineering processes They are applied to plants.

As one of such methods, it is known the technique of inserting a DNA fragment (DNA = acid deoxyribunucleic) originated from an intron in a site located on the head side with respect to the foreign gene. By example, the Printed Copy of the Parts of the Patent Application Japanese (Kokai) (Kokai = unexamined) No. 3-103182 Distributed to the Public describes that the expression of a foreign gene it is promoted by inserting a DNA fragment originated from a castor catalase gene intron (CAT-1) in a site located on the head side with respect to the foreign gene, and expressing the foreign gene. Similar phenomena have been reported. for various DNA fragments originated from introns.

Although introns have been used for the purpose of promoting the expression of foreign genes, the use of a plurality of introns has not become popular, and the advantageous effect thereof has not been recognized. For example, although the first intron and the 6th intron of the corn alcohol dehydrogenase gene individually promote the expression of the gene, if these introns are linked the effect is less than that produced in the case in which 6th intron is used alone (Mascarenhas et al . Plant Mol. Biol., 15, 913-920 (1990)). Similarly, in cases where two third corn actin introns are linked, the effect is less than that produced in the case where only one intron is used (Luehrsen et al ., Mol. Gen. Genet. , 225, 81-93
(1991)).

Several methods for expressing a foreign gene are known in the art. Mascarenhas et al . (Plant Molecular Biology, vol. 15, (1990), pp. 913-920, "Intron-Mediated Enhancement of Heterologous Gene Expression in Maize") describes constructs in which introns 2 and 6 of the corn Adh1 gene were inserted into the 5 'side on the head side with respect to the CAT coding region.

Luehrsen et al ., (Mol. Gen. Genet., Vol. 225, (1991), pp. 81-93. "Intron Enhancement of Gene Expression and the Splicing Efficiency of Introns in Maize Cells") describes methods for the expression of human serum albumin (HSA) using vectors consisting of a β-lactoglobulin (BLG) promoter that activates HSA expression and comprises specific subsets of introns, driving the uses of certain specific subsets to higher HSA expression .

Hurwitz et al ., (Transgenic Research, vol. 3, no. 6, November 1, 1994, pp. 376-375), also describes a series of expression vectors each consisting of a BLG promoter that activates one of those of a variety of HSA minigenes or the entire gene.

Christensen et al ., (Plant Molecular Biology, vol. 18, (1992), pp. 675-689, "Maize Polyubiquitin Genes: Structure, Thermal Perturbation of Expression and Transcript Splicing and Promoter Activity Following Transfer Protoplasts by Electroporation"), describes the increased cDNA expression (cDNA = complementary DNA) of human lactoferrin using heterologous introns.

Kim et al ., (Journal of Biochemistry and Molecular Biology, vol. 1, no. 28, January 31, 1995, pp. 57-61), describes that the expression of a recombinant human lactoferrin in mouse HC11 mammary epithelial cells was achieved putting your cDNA under the control of the bovine β-casein gene. To improve the expression level of human lactoferrin in a cell culture system, two artificial introns were also introduced to construct expression vectors. An intron was a hybridization signal consisting of the intron I of bovine β-casein and the intron II of rabbit β-globin. The other intron was a DNA fragment that encompasses intron VIII of the β-casein gene
bovine

EP 0 342 926 (Lubrizol Genetics, Inc.) describes a promoter region of corn ubiquitin that It includes consensus elements of thermal shock and initiates and regulates transcription of the genes placed under your control.

Although the methods known in the which is inserted a DNA fragment originated from a intron are effective, expression promotion effects are a often insufficient. Thus, it is to be desired a method by which gene expression is promoted to a greater extent.

Exhibition of the invention

Consequently, an object of this invention is to provide a method to express foreign genes whereby foreign genes are expressed to a greater extent that by known methods, and provide recombinant vectors for it.

The present inventors have carried out intensive studies to discover that gene expression foreign is promoted to a much greater extent by inserting in one or several sites on the head side with respect to the foreign gene a plurality of DNA fragments that have originated from introns, are the same or different and are capable of promoting foreign gene expression when inserted into a site located in the head side with respect to the foreign gene, compared to the known methods in which a single fragment of DNA originated from an intron, having thereby achieved Carry out the present invention.

This means that the present invention provides a method that is to express a foreign gene and includes the steps of inserting said foreign gene in a site located on the side of tail with respect to a promoter, and expressing said foreign gene in a cell; said method being characterized by the fact that those of a plurality of DNA fragments that have originated from starting from introns, they are the same or different and are capable of promote the expression of foreign genes are inserted into one or several sites located on the head side with respect to said gene foreign and said foreign gene is expressed, comprising at least one of said plurality of DNA fragments originating from from introns the sequence indicated in SEQ ID NO: 1 in the Sequence Listing or a functional variant thereof.

The present invention also provides a vector recombinant comprising a promoter, a foreign gene inserted into a site located on the tail side with respect to said promoter, and a plurality of DNA fragments that have originated from of introns, they are the same or different and are capable of promoting expression of foreign genes, said DNA fragments being inserted into one or several sites on the head side with respect to said foreign gene, comprising at least one of said plurality of DNA fragments originated from introns the sequence indicated in SEQ ID NO: 1 in the List of Sequences or a functional variant thereof.

Through the present invention they have been provided methods for expressing foreign genes through which the foreign gene expression is promoted in much greater as in the case of known methods, as well as vectors recombinant for it. With the present invention, since it promotes the expression of foreign genes introduced by genetic engineering processes, hopefully the present invention will constitute a huge contribution to the field of genetic engineering.

The best way to carry out the invention

The method of the present invention is characterized by the step of inserting two or more DNA fragments that have been originated from introns and are capable of promote the expression of foreign genes in one or several sites in the head side with respect to a foreign gene to express, so Promote the expression of the foreign gene.

Here, the expression "DNA fragment that has been originated from an intron and has the effect of promoting the expression of foreign genes" means a DNA fragment that has originated from an intron and is capable of promoting expression of foreign genes in detectable degree compared to the case in which the foreign gene is expressed without inserting the DNA fragment originated from an intron. Several DNA fragments originating from introns of this type are known per se . Examples of such DNA fragments originating from introns include the first intron of the castor catalase gene (CAT-1) (Printed Copy of Parts of Japanese Patent Application (Kokai) No. 3-103182; Tanaka et al. Nucleic Acids Res. 18, 6767-6770 (1990)); the flavonol glycosyltransferase intron: UDP-glucose from corn (Callis et al ., Genes & Develop. 1, 1183-1200 (1987)); the first intron of corn alcohol dehydrogenase-1 (Callis et al ., Genes & Develop. 1, 1183-1200 (1987)); the second and sixth intron of corn alcohol dehydrogenase-1 (Mascarenhas et al ., Plant Mol. Biol. 15, 913-920 (1990)); the first intron of the "shrunken-1"("shrunk-1") of corn (Vasil et al ., Plant Physiol. 91, 1575-1579 (1989)); the first intron of the elongation factor EF-1 alpha from the translation of Arabidopsis thaliana; and the first rice actin intron (McElroy et al ., Plant Cell 2, 163-171 (1990)). It should be noted, however, that DNA fragments that are originated from introns and can be employed in the present invention are not limited to the above enumeration, and any DNA fragments that have originated from introns can be employed. and can promote the expression of foreign genes on the tail side with respect to them.

The present inventors discovered previously introns of the rice PLD gene comparing the nucleotide sequences of the cDNA and genomic DNA of the gene of the rice phospholipase D (PLD), discovered that one of these introns prominently promotes gene expression on the side of queue with respect to it, and submitted a request for related patent (PCT / JP96 / 00812). The sequence of Nucleotides of this intron are indicated in SEQ ID NO: 1 in the Sequence Listing. In the present invention this can be used DNA fragment originated from an intron and indicated in SEQ ID NO: 1. In addition, the intron sequence of the castor catalase gene, indicated in the ID SEC Nº: 2 in the Sequence List and the intron sequence that is presents in the ubiquitin gene of corn and has the sequence of nucleotides indicated in SEQ ID NO: 3.

It is perfectly known in the art that there are cases in which the physiological activity of a DNA sequence physiologically active is maintained even if added, inserted, deleted or substituted one or more nucleotides. In the present invention are included in the expression "fragments of DNA originated from introns "in the sense in which the it is used herein the DNA fragments resulting from such a modification of those described above and known DNA fragments originating from introns or from the sequence indicated in SEQ ID NO: 1 as such DNA fragments promote gene expression on the tail side With respect to them. This means that they are also included in the expression "DNA fragments originating from of introns "in the present invention the DNA fragments that they have the same nucleotide sequences as above mentioned and known DNA fragments originated from introns or the DNA fragment that has been originated from a intron and has the nucleotide sequence indicated in the ID SEC Nº: 1, except for the fact that they are added, deleted or substituted one or more nucleotides, which as such fragments of DNA promote the expression of a gene on the tail side with Regarding them. Here, a DNA fragment of this type modified and originated from an intron preferably has a homology of not less than 70%, and more preferably of not less than 90%, with respect to the original DNA fragment and originated from an intron. Similarly, the expression "functional variant" used in the drafting of claims means the DNA fragment that has the same nucleotide sequence as the original sequence except the fact that one or more are added, deleted or substituted nucleotides, and that as such a DNA fragment promotes expression of a gene on the tail side with respect to it and has preferably a homology of not less than 70%, and more preferably not less than 90%, with respect to the sequence original. For example, the expression "functional variant of the sequence indicated in SEQ ID NO: 1 "means the DNA fragment that has the same nucleotide sequence as the indicated in SEQ ID NO: 1 except for the fact that they are added, deleted or substituted one or more nucleotides, and that as such a DNA fragment promotes the expression of a gene on the side of tail with respect thereto and preferably has a homology of not less than 70%, and more preferably of not less than 90%, with respect to the original sequence.

Each of the DNA fragments above described can be easily prepared by the method Conventional polymerase chain reaction (PCR) post that the nucleotide sequence and the original source are known Of the same. In addition, those with sizes of no more than approximately 200 base pairs (bp) can be synthesized chemically The aforementioned DNA fragments originated from of introns and modified can be easily prepared by the method of site-specific mutagenesis or by chemical synthesis

In the method of the present invention, those of a plurality of DNA fragments originated from introns and previously mentioned are inserted in one or several sites in the head side with respect to the foreign gene to express, that is at one or several sites on the head side with respect to the region of transcription, and more preferably, at the 5 'end of the region of transcription. DNA fragments originated from introns can preferably be inserted between the promoter and the foreign gene. DNA fragments originated from introns can be inserted into a site immediately located in the head side with respect to the foreign gene to express, or there may be another sequence between the originated DNA fragments from introns and the foreign gene. The length of this sequence intermediate is not limited and is usually 0 to 1000 bp. He promoter and DNA fragments originated from introns they can be directly connected, or there may be another sequence in between. The length of this intermediate sequence does not It is limited and is usually 0 to 1000 bp.

In the method of the present invention is inserted a plurality of preferably 2 to 5, and more preferably 2 or 3, of the previously described DNA fragments originating from from introns. DNA fragments originated from introns to be inserted can have the same nucleotide sequence or well different nucleotide sequences. Those of the plurality of DNA fragments originating from introns may be directly connected, or there may be another sequence In between. The length of this intermediate sequence is not limited and is usually 0 to 1000 bp.

Any promoter that can be used as a promoter can express the foreign gene located on the tail side with Regarding it. A preferred example of the promoter is the promoter 35S, although the promoter is not limited to it.

The present invention also provides a vector recombinant to which the method of the present invention is applied which has been described above. This means that the The present invention provides a recombinant vector comprising a promoter, a foreign gene inserted into a tail side site with respect to the promoter, and a plurality of DNA fragments that have originated from introns, are the same or different, they are able to promote the expression of foreign genes and are inserted into one or several sites on the head side with respect to the foreign gene. A vector of this type can be obtained by inserting the previously described plurality of DNA fragments originated from introns and the foreign gene in an appropriate expression vector. The insertion can be done easily using appropriate restriction enzymes and linkers, if necessary, because the site nucleotide sequence is known of cloning the expression vector.

They are known in the art and are available commercially several expression vectors of this type. These expression vectors comprise at least one origin of replication for replication in the host cell, a promoter, a site of cloning that provides restriction sites to insert a gene foreign, and a selection marker such as a marker of drug resistance Such expression vectors comprise usually a terminator to end with stability the transcription and an SD sequence in cases where the cell Host is a bacterial cell. In the method of this invention any of these vectors of known expression.

The present inventors discovered that although a single intron sequence of the gene from is inserted into a foreign gene corn ubiquitin, which has the nucleotide sequence that is indicated in SEQ ID NO: 3 in the Sequence Listing, it is promoted Foreign gene expression. However, even in cases in those that use the sequence indicated in SEQ ID NO: 3, the effect is greater when a plurality of sequences is inserted, and the effect is especially marked when the sequence indicated in SEQ ID NO: 3 it is inserted together with the intron sequence of the PLD gene of rice, which is indicated in SEQ ID NO: 1.

Examples

The more specifically described below is invention by means of examples thereof. It should be noted, without However, that the present invention is not limited to the examples following.

Example one

In the rice PLD gene there is a first intron with a size of 173 bp in the region corresponding to the region not 5 'end coding of the mRNA (mRNA = ribonucleic acid messenger) (SEQ ID NO: 1, WO 95/09234). The intron was verified to determine its influence on gene expression in cells vegetables. 20imer primers were synthesized (5'-TCACCACCCGGTAAGCCCAG-3 ', 3'-CCCCCGCGTCCATCCCGCTC-5 '), each one of which contains a 10 nucleotide region originated from starting from an exon, and the polymerase chain reaction was carried out using rice genomic clone as a template. The Polymerase chain reaction (PCR) was carried out using a mixture of 50 pmoles of each of the primers, 200 µM of dATP, dCTP, dGTP and dTTP, 1 x PCR buffer (which is supplied commercially by TAKARA SHUZO) and 2.5 U of AmpliTap DNA polymerase (TAKARA SHUZO), with 50 µl being the total volume of the reaction mixture. The reaction was carried out according to thermal conditions indicated below, and the cycle was Repeated 30 times These conditions were, in a DNA THERMOCYCLER (commercially supplied by PARKIN ELMER CETUS), those of 94ºC for 1 minute, 40 ° C for 1 minute and 72 ° C for space of 2 minutes and 30 seconds.

The PCR product was subcloned into the PCR II vector (commercially supplied by INVITROGEN), and a fragment was cut with Eco RI. The fragment became blunt and was inserted into the Sma I site of pBI221 (a vector plasmid in which a β-glucuronidase gene (called "GUS" hereinafter)) is inserted into a tail side site with with respect to a 35S promoter (called "35S pro" hereafter)), which is commercially supplied by TOYOBO CO., LTD., to obtain a pBI221P vector (35S pro, intron of PLD, GUS). This plasmid was digested with Bam HI and the resulting fragments became blunt, then incorporation was carried out.
ration of the fragment described above to construct a pBI221PP vector (35S pro, intron of PLD x 2, GUS).

The pIG221 (which had the intron (SEQ ID NO: 2) of the castor and GUS catalase gene in the order mentioned, in a tail-side region with respect to the 35S pro) that is described in the Printed Copy of Parts of the Japanese Patent Application Distributed to the Public (Kokai) No. 3-103182 was digested with Xba I, and the resulting fragments became blunt, the insertion of the intron sequence of PLD described above was then carried out to construct a vector pIG221P (35S pro, intron of PLD, intron of Catalase, GUS).

Cultivated rice cells were prepared from the immature embryo of the Nihonbare variety of Japanese rice (Hiei et al ., The Plant Journal, 6, 271-282 (1994)), the gene described above was introduced into the cells according to a method reported (Shimamoto et al . Nature, 338, 274-276 (1989)), and β-glucuronidase (GUS) activities were measured. The results are indicated in Table 1.

As indicated in Table 1, by introduction of two same or different introns GUS activity it was markedly increased compared to cases in that a single intron was used. It was also shown that the DNA fragment that has the intron nucleotide sequence of PLD is the DNA fragment that produces the effect of promoting gene expression when two or more of the fragments of DNA

TABLE 1

Plasmid GUS activity None 7.2 pBI221 (35S pro, GUS) 14 pBI221P (35S pro, Intron of PLD, GUS) 180 pBI221PP (35S pro, Intron of PLD x 2, GUS) 430 pIG221 (35S pro, Intron of Catalase, GUS) 160 pIG221P (35S pro, Intron of PLD, Intron of Catalase, GUS) 680

Example 2

The effect of promoting the expression of foreign genes was also verified for the intron of the corn ubiquitin gene (Ubi-1: Christensen AH et al . Plant Mol. Biol., 18, 675-689 (1982)). Two kinds of vectors were used.

It was first constructed using the method indicated below a vector to examine the effect of intron when a single intron is inserted. What was done was to separate the region of the promoter and intron from the ubiquitin gene by cutting with Pst I, and the fragment obtained (SEQ ID NO: 3) was inserted into the Pst I site of the pUC18 vector. The intron region was separated by cutting with Bgl II and Bam HI, and the fragment obtained was inserted into the Bam HI site of the pBI221 vector to obtain a pBI221U vector (pro 35S, Ubiquitin intron, GUS).

It was then constructed using the method indicated below a vector to examine the effect of intron when a plurality of introns is inserted. What was done was to separate the intron region by cutting with Bgl II and Bam HI, and the fragment obtained was blunt, then inserting the resulting fragment into the Sma I site of the vector pBI221. The Bam HI site of this vector was blunted, and the previously described PLD intron was inserted there to obtain a pBI221PU vector (35S pro, PLD intron, Ubiquitin intron, GUS). Vectors were introduced into the protoplasts using the aforementioned method, and GUS activities were measured.

As indicated in Table 2, it was observed that when a single ubiquitin intron was used the GUS expression. A more marked promotional effect was observed at employ both the intron of PLD and the intron of ubiquitin, in comparison with the cases in which it was used individually each One of the introns.

TABLE 2

Plasmid Activity of GUS (pmol MU / min. / Mg of protein) None 3.3 pBI221 (35S pro, GUS) 13 pBI221 (35S pro, Intron of PLD, GUS) 100 pBI221 (35S pro, Ubiquitin Intron, GUS) 360 pBI221 (35S pro, Intron of PLD, Ubiquitin Intron, GUS) 780

SEQ ID NO: 1

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NAME: Japan Tobacco Inc.

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COUNTRY: Japan

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  TITLE OF THE INVENTION: Method for Expressing Foreign Genes and Vectors for it.

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APPLICATION NUMBER: 97926233.4

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SUBMISSION DATE: June 12, 1997

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6

Claims (12)

1. A method that is for expressing a foreign gene and comprises the steps of inserting said foreign gene at a tail side site with respect to a promoter and expressing said foreign gene in a cell; characterized by the fact that those of a plurality of DNA fragments that have been originated from introns, are the same or different and are capable of promoting the expression of foreign genes are inserted into one or several sites on the head side with with respect to said foreign gene and said foreign gene is expressed, at least one of said plurality of DNA fragments originating from introns comprising the sequence indicated in SEQ ID NO: 1 in the Sequence Listing or a variant functional of the same that has a homology of not less than 90% with respect to SEQ ID NO: 1. 2. Method according to claim 1, wherein said plurality of DNA fragments originating from introns comprises two sequences indicated in SEQ ID NO: 1 in the Sequence Listing or a functional variant thereof that has a homology of not less than 90% with respect to the SEQ ID Nº: 1. 3. Method according to claim 1, wherein at least one of said plurality of DNA fragments originating from introns comprises the sequence that is indicated in SEQ ID NO: 2 in the Sequence Listing or a functional variant thereof that has a homology of not less of 90% with respect to SEQ ID NO: 2. 4. Method according to claim 3, wherein said plurality of DNA fragments originating from introns comprise the sequence indicated in SEQ ID NO: 1 or a Functional variant thereof and the sequence indicated in the SEQ ID Nº: 2 or a functional variant thereof, having the variants functional a homology of not less than 90% with respect to the original sequence 5. Method according to claim 1, wherein at least one of said plurality of DNA fragments originating from introns comprises the sequence that is indicated in SEQ ID NO: 3 in the Sequence Listing or a functional variant thereof that has a homology of not less of 90% with respect to SEQ ID NO: 3. 6. Method according to claim 5, wherein said plurality of DNA fragments originating from introns comprise the sequence that is indicated in SEQ ID NO: 1 or a functional variant thereof and the sequence that is indicated in SEQ ID NO: 3 or a functional variant thereof, the functional variants having a homology of not less than one 90% with respect to the original sequence. 7. Recombinant vector comprising a promoter, a foreign gene inserted into a tail side site with respect to said promoter, and a plurality of DNA fragments that have been originated from introns, are the same or different, they are able to promote the expression of foreign genes and are inserted in one or several sites on the head side with with respect to said foreign gene, comprising at least one of those of said plurality of DNA fragments originating from introns the sequence that is indicated in SEQ ID NO: 1 in the List of Sequences or a functional variant thereof that has a homology of not less than 90% with respect to SEQ ID NO: one. 8. Recombinant vector according to claim 7, wherein said plurality of DNA fragments originating from starting from introns are the two sequences indicated in the ID SEC Nº: 1 in the Sequence List or a functional variant of they have a homology of not less than 90% with regarding SEQ ID NO: 1. 9. Recombinant vector according to claim 7, wherein at least one of said plurality of fragments of DNA originating from introns comprises the sequence that is indicated in SEQ ID NO: 2 in the Sequence Listing or a functional variant thereof that has a homology of not less of 90% with respect to SEQ ID NO: 2. 10. Recombinant vector according to claim 9, wherein said plurality of DNA fragments originating from starting from introns comprises the sequence indicated in the ID SEC Nº: 1 or a functional variant thereof and the sequence that is indicated in SEQ ID NO: 2 or a functional variant of the same, the functional variants having a homology of not less of 90% with respect to the original sequence. 11. Recombinant vector according to claim 7, wherein at least one of said plurality of fragments of DNA originating from introns comprises the sequence that is indicated in SEQ ID NO: 3 in the Sequence Listing or a functional variant thereof that has a homology of not less of 90% with respect to SEQ ID NO: 3. 12. Recombinant vector according to claim 11, wherein said plurality of DNA fragments originating from starting from introns comprises the sequence indicated in the ID SEC Nº: 1 or a functional variant thereof and the sequence that is indicated in SEQ ID NO: 3 or a functional variant of the same, the functional variants having a homology of not less of 90% with respect to the original sequence.